European Journal of Human Genetics (2002) 11, 155 – 162 ª 2002 Nature Publishing Group All rights reserved 1018 – 4813/02 $25.00 www.nature.com/ejhg ARTICLE Identification and characterisation of the retinitis pigmentosa 1-like1 gene (RP1L1): a novel candidate for retinal degenerations Ivan Conte1,2, Marta Lestingi1, Anneke den Hollander3, Giovanna Alfano1, Carmela Ziviello1, Mariarosaria Pugliese1, Diego Circolo1, Cristina Caccioppoli1, Alfredo Ciccodicola*,1,2 and Sandro Banfi1 1Telethon Institute of Genetics and Medicine (TIGEM), Via Pietro Castellino 111, 80131 Naples, Italy; 2Institute of Genetics and Biophysics ‘A. Buzzati-Traverso’ (IGB), CNR, Naples, Italy; 3Department of Human Genetics, University Medical Center Nijmegen, Nijmegen, The Netherlands Retinitis pigmentosa (RP) is the most common form of inherited retinopathy, with an approximate incidence of 1 in 3700 individuals worldwide. Mutations in the retinitis pigmentosa 1 (RP1) gene are responsible for about 5 – 10% cases of autosomal dominant RP. The RP1 gene is specifically expressed in the photoreceptor layers of the postnatal retina and encodes a predicted protein characterised by the presence of two doublecortin (DC) domains, known to be implicated in microtubule binding. We identified and characterised, both in human and in mouse, a novel mammalian gene, termed Retinitis Pigmentosa1-like1 (RP1L1), because of its significant sequence similarity to the RP1 gene product. The sequence homology between RP1 and RP1L1 was found to be mostly restricted to the DC domains and to the N-terminal region, including the first 350 amino acids. The RP1L1 gene was also found to be conserved in distant vertebrates, since we identified a homologue in Fugu rubripes (pufferfish). Similar to RP1, RP1L1 expression is restricted to the postnatal retina, as determined by semiquantitative reverse transcriptase-PCR and Northern analysis. The retina-specific expression and the sequence similarity to RP1 render RP1L1 a potential candidate for inherited retinal disorders. European Journal of Human Genetics (2002) 11, 155 – 162. doi:10.1038/sj.ejhg.5200942 Keywords: RP1L1; RP1; retinitis pigmentosa; DC domain Introduction geneous and is inherited in an autosomal recessive, autoso- Retinitis pigmentosa (RP; MIM 268000) is a group of inher- mal dominant or X-linked fashion. However, in the ited retinal degeneration disorders, affecting 1 in 3700 majority of cases (about 50 – 60% in the Caucasian popula- individuals in the world population (http://www.ncbi.nlm. tion) it is impossible to determine the pattern of nih.gov/Omim/). It is characterised by night blindness, inheritance by the absence of family history. To date, muta- progressive loss of the peripheral visual field, bone tions in over thirty genes have been found to have a spicule-like pigmentary deposits and abnormal electroreti- causative role in RP, but a large number of RP genes have nograms (ERGs). This condition is genetically hetero- not yet been identified (http://www.sph.uth.tmc.edu/ Retnet/). *Correspondence: A Ciccodicola, Institute of Genetics and Biophysics ‘A. The retinitis pigmentosa 1 (RP1) gene plays a role in the 1,2 Buzzati-Traverso’ (IGB), CNR, and Telethon Institute of Genetics and pathogenesis of 5 – 10% of autosomal dominant RP. RP1 Medicine (TIGEM), Naples, Italy. Tel.: +39-081-6132259; is a photoreceptor-specific gene located on chromosome Fax: +39-081-6132352; 8q12, and encodes a predicted protein sharing, in its N- E-mail: [email protected] or [email protected] Received 26 September 2002; revised 19 November 2002; accepted 27 terminal region, significant sequence homology with a November 2002 domain of the human doublecortin protein (DC domain) Identification of the RP1L1 gene IConteet al 156 known to interact with microtubules.1,3,4 Recently, immu- the following: o5F, 5’-CTC CAG CTT TCC GCT CAG CC-3’; nofluorescence analysis showed that the RP1 protein is o5R, 5’-AGC CTC CAG CAC CGG CCT C-3’. localised in the connecting cilia of rod and cone photore- To identify the full-length of the RP1L1 gene, we ceptors, which suggests that this protein plays a role in performed 5’ and 3’ RACE – PCR on human retina Mara- the transport of proteins or in the maintenance of cilial thon-Ready cDNA (Clontech) according to the protocol structure.5 This hypothesis is also strengthened by recent provided. A first round PCR was performed using the adap- studies carried out in mice with a targeted disruption of tor primer AP1 (Clontech) and the gene-specific primers o5/ the Rp1 gene. In these mutant animals, photoreceptor cell 5RACE/F (5’-GTG GGA TGC AGG GAA GTC TTT GGC CGA layers undergo progressive degeneration and disorganisa- G-3’) and o5/3RACE/R (5’-ATG AGG TGA GTG CCA ACA tion. These abnormalities are preceded by mislocalisation GAA GCC AAG GAG-3’) for 5’ and 3’ RACE respectively. A of rhodopsin in inner segments and cell bodies of rods.6 nested PCR was then carried out using the adaptor primer We screened public EST databases to identify, catalogue AP2 and the specific primers o5/5RACE/nF (5’-AGC CAG and characterise novel retina-specific genes (manuscript in GAC AGT GCC AGC CCA G-3’) and o5/3RACE/nR (5’-AAA preparation). The highly specialised function of the retina GCC GTC TGC CCT GCC CAC TGC-3’) for 5’ and 3’ RACE, is likely to require a large number of genes expressed speci- respectively. We cloned the resulting PCR products in a fically or predominantly in this tissue. Such genes may play TOPO TA cloning vector (Invitrogen), according to the a critical role for the function of the retina and, when manufacturer’s instructions, and directly sequenced them. defective, may cause or predispose to retinal disease. In For analysis of polymorphisms in the human RP1L1 the course of this project, we identified a novel retina-speci- sequence, genomic DNA was extracted from peripheral fic gene, retinitis pigmentosa 1-like 1 (RP1L1), and its blood leukocytes using standard lysis/phenol extraction murine homologue Rp1l1, showing significant sequence protocols. PCR analysis was performed on 20 normal indivi- similarity to the RP1 gene. We performed a detailed study duals using oligonucleotide primers RP1L1/4-9F (5’-TCG of the expression of RP1L1 gene using both Northern-blot AAC CTG GAG CAG TTA GC-3’) and RP1L1/4-9R/5’-AGC and semi-quantitative RT – PCR analyses on different CTC TCC TTG CAG TCC TC-3’). PCR reactions were human tissues. We demonstrate that RP1L1 expression is performed with AmpliTaq Gold (Applied Biosystem) using restricted to the retina, suggesting that this gene may be a ABI 9700 automatic thermal cycler under the following involved in the pathogenesis of retinal degenerations. conditions: 958C for 10 min; then 35 cycles of 948C for 1 min, 598C for 1 min, and 728C for 2 min; and a final step Materials and methods of 728C for 10 min. PCR products were purified and PCR analysis sequenced as described above. For semi-quantitative RT – PCR, total RNA from seven human tissues (brain, liver, lung, skeletal muscle, placenta, Expression studies heart, and kidney) was purchased from Clontech. Total Northern-blot analysis Probe synthesis was obtained by RNA from retina, foetal eye, RPE/choroid, foetal cochlea PCR amplification on genomic DNA using primer pairs: h/ and RPE cell line, ARPE-19, was isolated with RNAzol B 5F (5’-GAG CCC TCA GGT CAG TCT AG-3’) and h/5R (5’- (Campro Scientific) or CsCl purification and treated with GCT CTC TGA CAC TTC TGG AC-3’) for the human gene, Dnase I (Gibco/BRL). Semi-quantitative RT – PCRs were and m/5F (5’-CAT CCA GTC TCC TCA GCT TC-3’) and m/ performed as described.7 A5mg aliquot of total brain, reti- 5R (5’-GTG GGA GTT GAT GAG TGA GC-3’) for the mouse na, and a retinoblastoma cell line (WERI-Rb) RNA, isolated gene. Human and mouse multiple-tissue Northern blots as previously described,8 was used for reverse transcription, (Clontech) were hybridised using (a-32P)dCTP-labelled which was carried out using random hexanucleotide prim- probes according to the protocol provided. The hybridised ing and Superscript II (Gibco BRL) in a 20 ml reaction, filters were washed once in 26SSC (300 mM NaCl, 30 mM according to the protocol provided by the manufacturer. sodium citrate, pH 7.5), 0.1% SDS at 658C, twice in PCR with RP1L1-specific primers was performed using 1 ml 16SSC, 0.1% SDS at 658C, and once in 0.56SSC, 0.1% of the reverse transcription reaction as template in a stan- SDS at 658C. dard PCR reaction with AmpliTaq Gold (Applied In situ hybridisation was performed using standard tech- Biosystem). In each experiment, a sample without reverse niques.9 The anti-sense DIG-labelled riboprobes were transcriptase was amplified under the same conditions as transcribed from three different probes corresponding to the reverse-transcribed RNA. PCR products were purified both the 5’ end and the 3’ end of the transcript. The ribop- from agarose gels by the Qiagen Gel extraction Kit and robes were prepared by the DIG RNA labelling Kit from directly sequenced on an automated sequencer (ABI 3100; Roche according to the protocol supplied. Hybridisation Applied Biosystem) using the ABI-PRISM big-dye terminator was performed on a minimum of three slides in at least five cycle sequencing ready reaction kit (Applied Biosystem). independent experiments. Tissue sections of 20 micron Oligonucleotide primers used in RT – PCR experiments were thickness were cut from CD1 mouse embryos at 14.5 days of gestation (E14.5) and adult eyes of different age (P40 – European Journal of Human Genetics Identification of the RP1L1 gene I Conte et al 157 80). For each eye, at least two series of sections were lation initiation codon,14 while the putative translation prepared.